Cancerous disease modifying antibodies

ABSTRACT

The present invention relates to a method for producing patient cancerous disease modifying antibodies using a novel paradigm of screening. By segregating the anti-cancer antibodies using cancer cell cytotoxicity as an end point, the process makes possible the production of anti-cancer antibodies for therapeutic and diagnostic purposes. The antibodies can be used in aid of staging and diagnosis of a cancer, and can be used to treat primary tumors and tumor metastases. The anti-cancer antibodies can be conjugated to toxins, enzymes, radioactive compounds, and hematogenous cells.

REFERENCE TO RELATED APPLICATIONS

This application claims benefit of the filing date of ProvisionalApplication 60/704,647, filed on Aug. 2, 2005, the contents of which areherein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the isolation and production of cancerousdisease modifying antibodies (CDMAB) and to the use of these CDMAB intherapeutic and diagnostic processes, optionally in combination with oneor more chemotherapeutic agents. The invention further relates tobinding assays which utilize the CDMAB of the instant invention.

BACKGROUND OF THE INVENTION

Monoclonal Antibodies as Cancer Therapy: Each individual who presentswith cancer is unique and has a cancer that is as different from othercancers as that person's identity. Despite this, current therapy treatsall patients with the same type of cancer, at the same stage, in thesame way. At least 30 percent of these patients will fail the first linetherapy, thus leading to further rounds of treatment and the increasedprobability of treatment failure, metastases, and ultimately, death. Asuperior approach to treatment would be the customization of therapy forthe particular individual. The only current therapy which lends itselfto customization is surgery. Chemotherapy and radiation treatment cannotbe tailored to the patient, and surgery by itself, in most cases isinadequate for producing cures.

With the advent of monoclonal antibodies, the possibility of developingmethods for customized therapy became more realistic since each antibodycan be directed to a single epitope. Furthermore, it is possible toproduce a combination of antibodies that are directed to theconstellation of epitopes that uniquely define a particular individual'stumor.

Having recognized that a significant difference between cancerous andnormal cells is that cancerous cells contain antigens that are specificto transformed cells, the scientific community has long held thatmonoclonal antibodies can be designed to specifically target transformedcells by binding specifically to these cancer antigens; thus giving riseto the belief that monoclonal antibodies can serve as “Magic Bullets” toeliminate cancer cells. However, it is now widely recognized that nosingle monoclonal antibody can serve in all instances of cancer, andthat monoclonal antibodies can be deployed, as a class, as targetedcancer treatments. Monoclonal antibodies isolated in accordance with theteachings of the instantly disclosed invention have been shown to modifythe cancerous disease process in a manner which is beneficial to thepatient, for example by reducing the tumor burden, and will variously bereferred to herein as cancerous disease modifying antibodies (CDMAB) or“anti-cancer” antibodies.

At the present time, the cancer patient usually has few options oftreatment. The regimented approach to cancer therapy has producedimprovements in global survival and morbidity rates. However, to theparticular individual, these improved statistics do not necessarilycorrelate with an improvement in their personal situation.

Thus, if a methodology was put forth which enabled the practitioner totreat each tumor independently of other patients in the same cohort,this would permit the unique approach of tailoring therapy to just thatone person. Such a course of therapy would, ideally, increase the rateof cures, and produce better outcomes, thereby satisfying a long-feltneed.

Historically, the use of polyclonal antibodies has been used withlimited success in the treatment of human cancers. Lymphomas andleukemias have been treated with human plasma, but there were fewprolonged remission or responses. Furthermore, there was a lack ofreproducibility and there was no additional benefit compared tochemotherapy. Solid tumors such as breast cancers, melanomas and renalcell carcinomas have also been treated with human blood, chimpanzeeserum, human plasma and horse serum with correspondingly unpredictableand ineffective results.

There have been many clinical trials of monoclonal antibodies for solidtumors. In the 1980s there were at least four clinical trials for humanbreast cancer which produced only one responder from at least 47patients using antibodies against specific antigens or based on tissueselectivity. It was not until 1998 that there was a successful clinicaltrial using a humanized anti-Her2/neu antibody (Herceptin®) incombination with cisplatin. In this trial 37 patients were assessed forresponses of which about a quarter had a partial response rate and anadditional quarter had minor or stable disease progression. The mediantime to progression among the responders was 8.4 months with medianresponse duration of 5.3 months.

Herceptin® was approved in 1998 for first line use in combination withTaxol®. Clinical study results showed an increase in the median time todisease progression for those who received antibody therapy plus Taxol®(6.9 months) in comparison to the group that received Taxol® alone (3.0months). There was also a slight increase in median survival; 22 versus18 months for the Herceptin® plus Taxol® treatment arm versus theTaxol(® treatment alone arm. In addition, there was an increase in thenumber of both complete (8 versus 2 percent) and partial responders (34versus 15 percent) in the antibody plus Taxol® combination group incomparison to Taxol® alone. However, treatment with Herceptin® andTaxol® led to a higher incidence of cardiotoxicity in comparison toTaxol® treatment alone (13 versus 1 percent respectively). Also,Herceptin® therapy was only effective for patients who over express (asdetermined through immunohistochemistry (IHC) analysis) the humanepidermal growth factor receptor 2 (Her2/neu), a receptor, whichcurrently has no known function or biologically important ligand;approximately 25 percent of patients who have metastatic breast cancer.Therefore, there is still a large unmet need for patients with breastcancer. Even those who can benefit from Herceptin® treatment would stillrequire chemotherapy and consequently would still have to deal with, atleast to some degree, the side effects of this kind of treatment.

The clinical trials investigating colorectal cancer involve antibodiesagainst both glycoprotein and glycolipid targets. Antibodies such as17-1A, which has some specificity for adenocarcinomas, has undergonePhase 2 clinical trials in over 60 patients with only 1 patient having apartial response. In other trials, use of 17-1A produced only 1 completeresponse and 2 minor responses among 52 patients in protocols usingadditional cyclophosphamide. To date, Phase III clinical trials of 17-1Ahave not demonstrated improved efficacy as adjuvant therapy for stageIII colon cancer. The use of a humanized murine monoclonal antibodyinitially approved for imaging also did not produce tumor regression.

Only recently have there been any positive results from colorectalcancer clinical studies with the use of monoclonal antibodies. In 2004,ERBITUX® was approved for the second line treatment of patients withEGFR-expressing metastatic colorectal cancer who are refractory toirinotecan-based chemotherapy. Results from both a two-arm Phase IIclinical study and a single arm study showed that ERBITUX® incombination with irinotecan had a response rate of 23 and 15 percentrespectively with a median time to disease progression of 4.1 and 6.5months respectively. Results from the same two-arm Phase II clinicalstudy and another single arm study showed that treatment with ERBITUX®alone resulted in an 11 and 9 percent response rate respectively with amedian time to disease progression of 1.5 and 4.2 months respectively.

Consequently in both Switzerland and the United States, ERBITUX®treatment in combination with irinotecan, and in the United States,ERBITUX® treatment alone, has been approved as a second line treatmentof colon cancer patients who have failed first line irinotecan therapy.Therefore, like Herceptin®, treatment in Switzerland is only approved asa combination of monoclonal antibody and chemotherapy. In addition,treatment in both Switzerland and the US is only approved for patientsas a second line therapy. Also, in 2004, AVASTIN® was approved for usein combination with intravenous 5-fluorouracil-based chemotherapy as afirst line treatment of metastatic colorectal cancer. Phase III clinicalstudy results demonstrated a prolongation in the median survival ofpatients treated with AVASTIN® plus 5-fluorouracil compared to patientstreated with 5-fluourouracil alone (20 months versus 16 monthsrespectively). However, again like Herceptin® and ERBITUX®, treatment isonly approved as a combination of monoclonal antibody and chemotherapy.

There also continues to be poor results for lung, brain, ovarian,pancreatic, prostate, and stomach cancer. The most promising recentresults for non-small cell lung cancer came from a Phase II clinicaltrial where treatment involved a monoclonal antibody (SGN-15; dox-BR96,anti-Sialyl-LeX) conjugated to the cell-killing drug doxorubicin incombination with the chemotherapeutic agent TAXOTERE®. TAXOTERE® is theonly FDA approved chemotherapy for the second line treatment of lungcancer. Initial data indicate an improved overall survival compared toTAXOTERE® alone. Out of the 62 patients who were recruited for thestudy, two-thirds received SGN-15 in combination with TAXOTERE® whilethe remaining one-third received TAXOTERE® alone. For the patientsreceiving SGN-15 in combination with TAXOTERE®, median overall survivalwas 7.3 months in comparison to 5.9 months for patients receivingTAXOTERE® alone. Overall survival at 1 year and 18 months was 29 and 18percent respectively for patients receiving SNG-15 plus TAXOTERE®compared to 24 and 8 percent respectively for patients receivingTAXOTERE® alone. Further clinical trials are planned.

Preclinically, there has been some limited success in the use ofmonoclonal antibodies for melanoma. Very few of these antibodies havereached clinical trials and to date none have been approved ordemonstrated favorable results in Phase III clinical trials.

The discovery of new drugs to treat disease is hindered by the lack ofidentification of relevant targets among the products of 30,000 knowngenes that unambiguously contribute to disease pathogenesis. In oncologyresearch, potential drug targets are often selected simply due to thefact that they are over-expressed in tumor cells. Targets thusidentified are then screened for interaction with a multitude ofcompounds. In the case of potential antibody therapies, these candidatecompounds are usually derived from traditional methods of monoclonalantibody generation according to the fundamental principles laid down byKohler and Milstein (1975, Nature, 256, 495-497, Kohler and Milstein).Spleen cells are collected from mice immunized with antigen (e.g. wholecells, cell fractions, purified antigen) and fused with immortalizedhybridoma partners. The resulting hybridomas are screened and selectedfor secretion of antibodies which bind most avidly to the target. Manytherapeutic and diagnostic antibodies directed against cancer cells,including Herceptin® and RITUXIMAB, have been produced using thesemethods and selected on the basis of their affinity. The flaws in thisstrategy are two-fold. Firstly, the choice of appropriate targets fortherapeutic or diagnostic antibody binding is limited by the paucity ofknowledge surrounding tissue specific carcinogenic processes and theresulting simplistic methods, such as selection by overexpression, bywhich these targets are identified. Secondly, the assumption that thedrug molecule that binds to the receptor with the greatest affinityusually has the highest probability for initiating or inhibiting asignal may not always be the case.

Despite some progress with the treatment of breast and colon cancer, theidentification and development of efficacious antibody therapies, eitheras single agents or co-treatments, has been inadequate for all types ofcancer.

Prior Patents:

U.S. Pat. No. 5,750,102 discloses a process wherein cells from apatient's tumor are transfected with MHC genes which may be cloned fromcells or tissue from the patient. These transfected cells are then usedto vaccinate the patient.

U.S. Pat. No. 4,861,581 discloses a process comprising the steps ofobtaining monoclonal antibodies that are specific to an internalcellular component of neoplastic and normal cells of the mammal but notto external components, labeling the monoclonal antibody, contacting thelabeled antibody with tissue of a mammal that has received therapy tokill neoplastic cells, and determining the effectiveness of therapy bymeasuring the binding of the labeled antibody to the internal cellularcomponent of the degenerating neoplastic cells. In preparing antibodiesdirected to human intracellular antigens, the patentee recognizes thatmalignant cells represent a convenient source of such antigens.

U.S. Pat. No. 5,171,665 provides a novel antibody and method for itsproduction. Specifically, the patent teaches formation of a monoclonalantibody which has the property of binding strongly to a protein antigenassociated with human tumors, e.g. those of the colon and lung, whilebinding to normal cells to a much lesser degree.

U.S. Pat. No. 5,484,596 provides a method of cancer therapy comprisingsurgically removing tumor tissue from a human cancer patient, treatingthe tumor tissue to obtain tumor cells, irradiating the tumor cells tobe viable but non-tumorigenic, and using these cells to prepare avaccine for the patient capable of inhibiting recurrence of the primarytumor while simultaneously inhibiting metastases. The patent teaches thedevelopment of monoclonal antibodies which are reactive with surfaceantigens of tumor cells. As set forth at col. 4, lines 45 et seq., thepatentees utilize autochthonous tumor cells in the development ofmonoclonal antibodies expressing active specific immunotherapy in humanneoplasia.

U.S. Pat. No. 5,693,763 teaches a glycoprotein antigen characteristic ofhuman carcinomas and not dependent upon the epithelial tissue of origin.

U.S. Pat. No. 5,783,186 is drawn to Anti-Her2 antibodies which induceapoptosis in Her2 expressing cells, hybridoma cell lines producing theantibodies, methods of treating cancer using the antibodies andpharmaceutical compositions including said antibodies.

U.S. Pat. No. 5,849,876 describes new hybridoma cell lines for theproduction of monoclonal antibodies to mucin antigens purified fromtumor and non-tumor tissue sources.

U.S. Pat. No. 5,869,268 is drawn to a method for generating a humanlymphocyte producing an antibody specific to a desired antigen, a methodfor producing a monoclonal antibody, as well as monoclonal antibodiesproduced by the method. The patent is particularly drawn to theproduction of an anti-HD human monoclonal antibody useful for thediagnosis and treatment of cancers.

U.S. Pat. No. 5,869,045 relates to antibodies, antibody fragments,antibody conjugates and single-chain immunotoxins reactive with humancarcinoma cells. The mechanism by which these antibodies function istwo-fold, in that the molecules are reactive with cell membrane antigenspresent on the surface of human carcinomas, and further in that theantibodies have the ability to internalize within the carcinoma cells,subsequent to binding, making them especially useful for formingantibody-drug and antibody-toxin conjugates. In their unmodified formthe antibodies also manifest cytotoxic properties at specificconcentrations.

U.S. Pat. No. 5,780,033 discloses the use of autoantibodies for tumortherapy and prophylaxis. However, this antibody is an antinuclearautoantibody from an aged mammal. In this case, the autoantibody is saidto be one type of natural antibody found in the immune system. Becausethe autoantibody comes from “an aged mammal”, there is no requirementthat the autoantibody actually comes from the patient being treated. Inaddition the patent discloses natural and monoclonal antinuclearautoantibody from an aged mammal, and a hybridoma cell line producing amonoclonal antinuclear autoantibody.

SUMMARY OF THE INVENTION

This application utilizes methodology for producing patient specificanti-cancer antibodies taught in the U.S. Pat. No. 6,180,357 patent forisolating hybridoma cell lines which encode for cancerous diseasemodifying monoclonal antibodies. These antibodies can be madespecifically for one tumor and thus make possible the customization ofcancer therapy. Within the context of this application, anti-cancerantibodies having either cell-killing (cytotoxic) or cell-growthinhibiting (cytostatic) properties will hereafter be referred to ascytotoxic. These antibodies can be used in aid of staging and diagnosisof a cancer, and can be used to treat tumor metastases. These antibodiescan also be used for the prevention of cancer by way of prophylactictreatment. Unlike antibodies generated according to traditional drugdiscovery paradigms, antibodies generated in this way may targetmolecules and pathways not previously shown to be integral to the growthand/or survival of malignant tissue. Furthermore, the binding affinitiesof these antibodies are suited to requirements for initiation of thecytotoxic events that may not be amenable to stronger affinityinteractions. Also, it is within the purview of this invention toconjugate standard chemotherapeutic modalities, e.g. radionuclides, withthe CDMAB of the instant invention, thereby focusing the use of saidchemotherapeutics. The CDMAB can also be conjugated to toxins, cytotoxicmoieties, enzymes e.g. biotin conjugated enzymes, or hematogenous cells,thereby forming an antibody conjugate.

The prospect of individualized anti-cancer treatment will bring about achange in the way a patient is managed. A likely clinical scenario isthat a tumor sample is obtained at the time of presentation, and banked.From this sample, the tumor can be typed from a panel of pre-existingcancerous disease modifying antibodies. The patient will beconventionally staged but the available antibodies can be of use infurther staging the patient. The patient can be treated immediately withthe existing antibodies, and a panel of antibodies specific to the tumorcan be produced either using the methods outlined herein or through theuse of phage display libraries in conjunction with the screening methodsherein disclosed. All the antibodies generated will be added to thelibrary of anti-cancer antibodies since there is a possibility thatother tumors can bear some of the same epitopes as the one that is beingtreated. The antibodies produced according to this method may be usefulto treat cancerous disease in any number of patients who have cancersthat bind to these antibodies.

In addition to anti-cancer antibodies, the patient can elect to receivethe currently recommended therapies as part of a multi-modal regimen oftreatment. The fact that the antibodies isolated via the presentmethodology are relatively non-toxic to non-cancerous cells allows forcombinations of antibodies at high doses to be used, either alone, or inconjunction with conventional therapy. The high therapeutic index willalso permit re-treatment on a short time scale that should decrease thelikelihood of emergence of treatment resistant cells.

If the patient is refractory to the initial course of therapy ormetastases develop, the process of generating specific antibodies to thetumor can be repeated for re-treatment. Furthermore, the anti-cancerantibodies can be conjugated to red blood cells obtained from thatpatient and re-infused for treatment of metastases. There have been feweffective treatments for metastatic cancer and metastases usuallyportend a poor outcome resulting in death. However, metastatic cancersare usually well vascularized and the delivery of anti-cancer antibodiesby red blood cells can have the effect of concentrating the antibodiesat the site of the tumor. Even prior to metastases, most cancer cellsare dependent on the host's blood supply for their survival and ananti-cancer antibody conjugated to red blood cells can be effectiveagainst in situ tumors as well. Alternatively, the antibodies may beconjugated to other hematogenous cells, e.g. lymphocytes, macrophages,monocytes, natural killer cells, etc.

There are five classes of antibodies and each is associated with afunction that is conferred by its heavy chain. It is generally thoughtthat cancer cell killing by naked antibodies are mediated either throughantibody dependent cellular cytotoxicity or complement dependentcytotoxicity. For example murine IgM and IgG2a antibodies can activatehuman complement by binding the C-1 component of the complement systemthereby activating the classical pathway of complement activation whichcan lead to tumor lysis. For human antibodies the most effectivecomplement activating antibodies are generally IgM and IgG1. Murineantibodies of the IgG2a and IgG3 isotype are effective at recruitingcytotoxic cells that have Fc receptors which will lead to cell killingby monocytes, macrophages, granulocytes and certain lymphocytes. Humanantibodies of both the IgG1 and IgG3 isotype mediate ADCC.

Another possible mechanism of antibody mediated cancer killing may bethrough the use of antibodies that function to catalyze the hydrolysisof various chemical bonds in the cell membrane and its associatedglycoproteins or glycolipids, so-called catalytic antibodies.

There are three additional mechanisms of antibody-mediated cancer cellkilling. The first is the use of antibodies as a vaccine to induce thebody to produce an immune response against the putative antigen thatresides on the cancer cell. The second is the use of antibodies totarget growth receptors and interfere with their function or to downregulate that receptor so that its function is effectively lost. Thethird is the effect of such antibodies on direct ligation of cellsurface moieties that may lead to direct cell death, such as ligation ofdeath receptors such as TRAIL R1 or TRAIL R2, or integrin molecules suchas alpha V beta 3 and the like.

The clinical utility of a cancer drug is based on the benefit of thedrug under an acceptable risk profile to the patient. In cancer therapysurvival has generally been the most sought after benefit, however thereare a number of other well-recognized benefits in addition to prolonginglife. These other benefits, where treatment does not adversely affectsurvival, include symptom palliation, protection against adverse events,prolongation in time to recurrence or disease-free survival, andprolongation in time to progression. These criteria are generallyaccepted and regulatory bodies such as the U.S. Food and DrugAdministration (F.D.A.) approve drugs that produce these benefits(Hirschfeld et al. Critical Reviews in Oncology/Hematolgy 42:137-1432002). In addition to these criteria it is well recognized that thereare other endpoints that may presage these types of benefits. In part,the accelerated approval process granted by the U.S. F.D.A. acknowledgesthat there are surrogates that will likely predict patient benefit. Asof year-end (2003), there have been sixteen drugs approved under thisprocess, and of these, four have gone on to full approval, i.e.,follow-up studies have demonstrated direct patient benefit as predictedby surrogate endpoints. One important endpoint for determining drugeffects in solid tumors is the assessment of tumor burden by measuringresponse to treatment (Therasse et al. Journal of the National CancerInstitute 92(3):205-216 2000). The clinical criteria (RECIST criteria)for such evaluation have been promulgated by Response EvaluationCriteria in Solid Tumors Working Group, a group of international expertsin cancer. Drugs with a demonstrated effect on tumor burden, as shown byobjective responses according to RECIST criteria, in comparison to theappropriate control group tend to, ultimately, produce direct patientbenefit. In the pre-clinical setting tumor burden is generally morestraightforward to assess and document. In that pre-clinical studies canbe translated to the clinical setting, drugs that produce prolongedsurvival in pre-clinical models have the greatest anticipated clinicalutility. Analogous to producing positive responses to clinicaltreatment, drugs that reduce tumor burden in the pre-clinical settingmay also have significant direct impact on the disease. Althoughprolongation of survival is the most sought after clinical outcome fromcancer drug treatment, there are other benefits that have clinicalutility and it is clear that tumor burden reduction, which may correlateto a delay in disease progression, extended survival or both, can alsolead to direct benefits and have clinical impact (Eckhardt et al.Developmental Therapeutics: Successes and Failures of Clinical TrialDesigns of Targeted Compounds; ASCO Educational Book, 39^(th) AnnualMeeting, 2003, pages 209-219).

The present invention describes the development and use of AR59A269.5identified by its effect in a cytotoxic assay and in an animal model ofhuman cancer. This invention describes reagents that bind specificallyto an epitope or epitopes present on the target molecule, and that alsohave in vitro cytotoxic properties, as a naked antibody, againstmalignant tumor cells but not normal cells, and which also directlymediate, as a naked antibody, inhibition of tumor growth. A furtheradvance is of the use of anti-cancer antibodies such as this to targettumors expressing cognate antigen markers to achieve tumor growthinhibition, and other positive endpoints of cancer treatment.

In all, this invention teaches the use of the AR59A269.5 antigen as atarget for a therapeutic agent, that when administered can reduce thetumor burden of a cancer expressing the antigen in a mammal. Thisinvention also teaches the use of CDMAB (AR59A269.5), and theirderivatives, and antigen binding fragments thereof, and cellularcytotoxicity inducing ligands thereof, to target their antigen to reducethe tumor burden of a cancer expressing the antigen in a mammal.Furthermore, this invention also teaches the use of detecting theAR59A269.5 antigen in cancerous cells that can be useful for thediagnosis, prediction of therapy, and prognosis of mammals bearingtumors that express this antigen.

Accordingly, it is an objective of the invention to utilize a method forproducing cancerous disease modifying antibodies (CDMAB) raised againstcancerous cells derived from a particular individual, or one or moreparticular cancer cell lines, which CDMAB are cytotoxic with respect tocancer cells while simultaneously being relatively non-toxic tonon-cancerous cells, in order to isolate hybridoma cell lines and thecorresponding isolated monoclonal antibodies and antigen bindingfragments thereof for which said hybridoma cell lines are encoded.

It is an additional objective of the invention to teach cancerousdisease modifying antibodies, ligands and antigen binding fragmentsthereof.

It is a further objective of the instant invention to produce cancerousdisease modifying antibodies whose cytotoxicity is mediated throughantibody dependent cellular toxicity.

It is yet an additional objective of the instant invention to producecancerous disease modifying antibodies whose cytotoxicity is mediatedthrough complement dependent cellular toxicity.

It is still a further objective of the instant invention to producecancerous disease modifying antibodies whose cytotoxicity is a functionof their ability to catalyze hydrolysis of cellular chemical bonds.

A still further objective of the instant invention is to producecancerous disease modifying antibodies which are useful for in a bindingassay for diagnosis, prognosis, and monitoring of cancer.

Other objects and advantages of this invention will become apparent fromthe following description wherein are set forth, by way of illustrationand example, certain embodiments of this invention.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 compares the percentage cytotoxicity and binding levels of thehybridoma supernatants against cell lines MDA-MB-231, OVCAR-3, SW1116,Lovo and CCD-27sk.

FIG. 2 represents binding of AR59A269.5 and the anti-EGFR control tocancer and normal cell lines. The data is tabulated to present the meanfluorescence intensity as a fold increase above isotype control.

FIG. 3 includes representative FACS histograms of AR59A269.5 andanti-EGFR antibodies directed against several cancer and non-cancer celllines.

FIG. 4 demonstrates the effect of AR59A269.5 on tumor growth in aprophylactic Lovo colon cancer model. The vertical lines indicate theperiod during which the antibody was administered. Data points representthe mean +/−SEM.

FIG. 5 demonstrates the effect of AR59A269.5 on body weight in aprophylactic Lovo colon cancer model. Data points represent the mean+/−SEM.

FIG. 6 demonstrates the effect of AR59A269.5 on tumor growth in aprophylactic DLD-1 colon cancer model. The vertical lines indicate theperiod during which the antibody was administered. Data points representthe mean +/−SEM.

FIG. 7 demonstrates the effect of AR59A269.5 on body weight in aprophylactic DLD-1 colon cancer model. Data points represent the mean+/−SEM.

FIG. 8 tabulates an IHC comparison of AR59A269.5 versus positive andnegative controls on human xenograft tumor tissue.

FIGS. 9A, 9B, 9C & 9D are representative micrographs showing the bindingpattern with AR59A269.5 on breast MDA-MB-231 (A) or colon SW1116 (B)xenograft tumor tissue or the buffer control on breast MDA-MB-231 (C) orcolon SW1116 (D) xenograft tumor tissue. AR59A269.5 displayed positivestaining for the tumor cells. Magnification is 200×.

DETAILED DESCRIPTION OF THE INVENTION

In general, the following words or phrases have the indicated definitionwhen used in the summary, description, examples, and claims.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies, de-immunized, murine,chimerized or humanized antibodies), antibody compositions withpolyepitopic specificity, single-chain antibodies, immunoconjugates andfragments of antibodies (see below).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma (murine orhuman) method first described by Kohler et al., Nature, 256:495 (1975),or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567). The “monoclonal antibodies” may also be isolated from phageantibody libraries using the techniques described in Clackson et al.,Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597(1991), for example.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include less than full length antibodies,Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules; single-chain antibodies, single domainantibody molecules, fusion proteins, recombinant proteins andmultispecific antibodies formed from antibody fragment(s).

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (C_(L)) and heavy chainconstant domains, C_(H)1, C_(H)2 and C_(H)3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variant thereof. Preferably, the intactantibody has one or more effector functions.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five-major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgG2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called a, d, e, ?, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express Fc?RIII only, whereas monocytes express Fc?RI, Fc?RII andFc?RIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFc?RIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fe region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the Fc?RI, Fc?RII,and Fc? RIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. Fc?RII receptors include Fc?RIIA (an“activating receptor”) and Fc?RIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor Fc?RIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor Fc?RIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (seereview M. in Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., Eur. J Immunol. 24:2429 (1994)).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996) may be performed.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the >sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. pp 15-17; 48-53 (1991)).The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody dependent cellularcytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md. pp15-17; 48-53 (1991)) and/or those residues from a “hypervariable loop”(e.g. residues 2632 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96 -101 (H3) in the heavychain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917(1987)). “Framework Region” or “FR” residues are those variable domainresidues other than the hypervariable region residues as herein defined.Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site. The Fab fragment alsocontains the constant domain of the light chain and the first constantdomain (CH I) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear at least onefree thiol group. F(ab′)₂ antibody fragments originally were produced aspairs of Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (?) andlambda (?), based on the amino acid sequences of their constant domains.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(V_(H)) connected to a variable light domain (V_(L)) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

An antibody “which binds” an antigen of interest is one capable ofbinding that antigen with sufficient affinity such that the antibody isuseful as a therapeutic or diagnostic agent in targeting a cellexpressing the antigen. Where the antibody is one which binds theantigenic moiety it will usually preferentially bind that antigenicmoiety as opposed to other receptors, and does not include incidentalbinding such as non-specific Fc contact, or binding topost-translational modifications common to other antigens and may be onewhich does not significantly cross-react with other proteins. Methods,for the detection of an antibody that binds an antigen of interest, arewell known in the art and can include but are not limited to assays suchas FACS, cell ELISA and Western blot.

As used herein, the expressions “cell”, “cell line”, and “cell culture”are used interchangeably, and all such designations include progeny. Itis also understood that all progeny may not be precisely identical inDNA content, due to deliberate or inadvertent mutations. Mutant progenythat have the same function or biological activity as screened for inthe originally transformed cell are included. It will be clear from thecontext where distinct designations are intended.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.Hence, the mammal to be treated herein may have been diagnosed as havingthe disorder or may be predisposed or susceptible to the disorder.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth or death. Examples of cancer include, but arenot limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia orlymphoid malignancies. More particular examples of such cancers includesquamous cell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, as well as head and neckcancer.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotociri, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, camomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2?-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Aventis, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and anti-androgens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, mice, SCID or nude mice or strains of mice,domestic and farm animals, and zoo, sports, or pet animals, such assheep, dogs, horses, cats, cows, etc. Preferably, the mammal herein ishuman.

“Oligonucleotides” are short-length, single-or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(such as phosphotriester, phosphite, or phosphoramidite chemistry, usingsolid phase techniques such as described in EP 266,032, published 4 May1988, or via deoxynucleoside H-phosphonate intermediates as described byFroehler et al., Nucl. Acids Res., 14:5399-5407, 1986. They are thenpurified on polyacrylamide gels.

“Chimeric” antibodies are immunoglobulins in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567 and Morrison et al, Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from thecomplementarity determining regions (CDRs) of the recipient antibody arereplaced by residues from the CDRs of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human FR residues. Furthermore, the humanized antibody may compriseresidues which are found neither in the recipient antibody nor in theimported CDR or FR sequences. These modifications are made to furtherrefine and optimize antibody performance. In general, the humanizedantibody will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of the CDRregions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR residues are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin.

“De-immunized” antibodies are immunoglobulins that are non-immunogenic,or less immunogenic, to a given species. De-immunization can be achievedthrough structural alterations to the antibody. Any de-immunizationtechnique known to those skilled in the art can be employed. Onesuitable technique for de-immunizing antibodies is described, forexample, in WO 00/34317 published Jun. 15, 2000.

An antibody which induces “apoptosis” is one which induces programmedcell death by any menas, illustrated by but not limited to binding ofannexin V, caspase activity, fragmentation of DNA, cell shrinkage,dilation of endoplasmic reticulum, cell fragmentation, and/or formationof membrane vesicles (called apoptotic bodies).

Throughout the instant specification, hybridoma cell lines, as well asthe isolated monoclonal antibodies which are produced therefrom, arealternatively referred to by their internal designation, AR59A269.5 orDepository Designation, IDAC 170505-01.

As used herein “antibody-ligand” includes a moiety which exhibitsbinding specificity for at least one epitope of the target antigen, andwhich may be an intact antibody molecule, antibody fragments, and anymolecule having at least an antigen-binding region or portion thereof(i.e., the variable portion of an antibody molecule), e.g., an Fvmolecule, Fab molecule, Fab′ molecule, F(ab′).sub.2 molecule, abispecific antibody, a fusion protein, or any genetically engineeredmolecule which specifically recognizes and binds at least one epitope ofthe antigen bound by the isolated monoclonal antibody produced by thehybridoma cell line designated as IDAC 170505-01 (the IDAC 170505-01antigen).

As used herein “cancerous disease modifiying antibodies” (CDMAB) refersto monoclonal antibodies which modify the cancerous disease process in amanner which is beneficial to the patient, for example by reducing tumorburden or prolonging survival of tumor bearing individuals, andantibody-ligands thereof.

As used herein “antigen-binding region” means a portion of the moleculewhich recognizes the target antigen.

As used herein “competitively inhibits” means being able to recognizeand bind a determinant site to which the monoclonal antibody produced bythe hybridoma cell line designated as IDAC 170505-01, (the IDAC170505-01 antibody) is directed using conventional reciprocal antibodycompetition assays. (Belanger L., Sylvestre C. and Dufour D. (1973),Enzyme linked immunoassay for alpha fetoprotein by competitive andsandwich procedures. Clinica Chimica Acta 48, 15).

As used herein “target antigen” is the IDAC 170505-01 antigen orportions thereof.

As used herein, an “immunoconjugate” means any molecule or CDMAB such asan antibody chemically or biologically linked to a cytotoxin, aradioactive agent, enzyme, toxin, an anti-tumor drug or a therapeuticagent. The antibody or CDMAB may be linked to the cytotoxin, radioactiveagent, anti-tumor drug or therapeutic agent at any location along themolecule so long as it is able to bind its target. Examples ofimmunoconjugates include antibody toxin chemical conjugates andantibody-toxin fusion proteins.

As used herein, a “fusion protein” means any chimeric protein wherein anantigen binding region is connected to a biologically active molecule,e.g., toxin, enzyme, or protein drug.

In order that the invention herein described may be more fullyunderstood, the following description is set forth.

The present invention provides CDMABs (i.e., IDAC 170505-01 CDMAB) whichspecifically recognize and bind the IDAC 170505-01 antigen.

The CDMAB of the isolated monoclonal antibody produced by the hybridomadeposited with the IDAC as accession number 170505-01 may be in any formas long as it has an antigen-binding region which competitively inhibitsthe immunospecific binding of the isolated monoclonal antibody producedby hybridoma IDAC 170505-01 to its target antigen. Thus, any recombinantproteins (e.g., fusion proteins wherein the antibody is combined with asecond protein such as a lymphokine or a tumor inhibitory growth factor)having the same binding specificity as the IDAC 170505-01 antibody fallwithin the scope of this invention.

In one embodiment of the invention, the CDMAB is the IDAC 170505-01antibody.

In other embodiments, the CDMAB is an antigen binding fragment which maybe a Fv molecule (such as a single-chain Fv molecule), a Fab molecule, aFab′ molecule, a F(ab′)2 molecule, a fusion protein, a bispecificantibody, a heteroantibody or any recombinant molecule having theantigen-binding region of the IDAC 170505-01antibody. The CDMAB of theinvention is directed to the epitope to which the IDAC 170505-01monoclonal antibody is directed.

The CDMAB of the invention may be modified, i.e., by amino acidmodifications within the molecule, so as to produce derivativemolecules. Chemical modification may also be possible.

Derivative molecules would retain the functional property of thepolypeptide, namely, the molecule having such substitutions will stillpermit the binding of the polypeptide to the IDAC 170505-01 antigen orportions thereof.

These amino acid substitutions include, but are not necessarily limitedto, amino acid substitutions known in the art as “conservative”.

For example, it is a well-established principle of protein chemistrythat certain amino acid substitutions, entitled “conservative amino acidsubstitutions,” can frequently be made in a protein without alteringeither the conformation or the function of the protein.

Such changes include substituting any of isoleucine (I), valine (V), andleucine (L) for any other of these hydrophobic amino acids; asparticacid (D) for glutamic acid (E) and vice versa; glutamine (Q) forasparagine (N) and vice versa; and serine (S) for threonine (T) and viceversa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanineand valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

Given an antibody, an individual ordinarily skilled in the art cangenerate a competitively inhibiting CDMAB, for example a competingantibody, which is one that recognizes the same epitope (Belanger L etal. Clinica Chimica Acta 48:15-18 (1973)). One method entails immunizingwith an immunogen that expresses the antigen recognized by the antibody.The sample may include but is not limited to tissues, isolatedprotein(s) or cell line(s). Resulting hybridomas could be screened usinga competition assay, which is one that identifies antibodies thatinhibit the binding of the test antibody, such as ELISA, FACS or Westernblotting. Another method could make use of phage display antibodylibraries and panning for antibodies that recognize at least one epitopeof said antigen (Rubinstein J L et al. Anal Biochem 314:294-300 (2003)).In either case, antibodies are selected based on their ability toout-compete the binding of the original labeled antibody to at least oneepitope of its target antigen. Such antibodies would therefore possessthe characteristic of recognizing at least one epitope of the antigen asthe original antibody.

EXAMPLE 1

The Hybridoma Production-Hybridoma Cell Line AR59A269.5

The hybridoma cell line AR59A269.5 was deposited, in accordance with theBudapest Treaty, with the International Depository Authority of Canada(IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street,Winnipeg, Manitoba, Canada, R3E 3R2, on May 17, 2005, under AccessionNumber 170505-01. In accordance with 37 CFR 1.808, the depositors assurethat all restrictions imposed on the availability to the public of thedeposited materials will be irrevocably removed upon the granting of apatent. The deposit will be replaced if the depository cannot dispenseviable samples.

To produce the hybridoma that produces the anti-cancer antibodyAR59A269.5, a single cell suspension of frozen human colon metastasis tothe liver tissue (Genomics Collaborative, Cambridge, Mass.) was preparedin PBS. IMMUNEASY™ (Qiagen, Venlo, Netherlands) adjuvant was preparedfor use by gentle mixing. Five to seven week old BALB/c mice wereimmunized by injecting subcutaneously, 2 million cells in 50 microlitersof the antigen-adjuvant. Recently prepared antigen-adjuvant was used toboost the immunized mice intraperitoneally, 2 and 5 weeks after theinitial immunization, with 2 million cells in 50 microliters. A spleenwas used for fusion three days after the last immunization. Thehybridomas were prepared by fusing the isolated splenocytes with NSO-1myeloma partners. The supernatants from the fusions were tested fromsubclones of the hybridomas.

To determine whether the antibodies secreted by the hybridoma cells areof the IgG or IgM isotype, an ELISA assay was employed. 100microliters/well of goat anti-mouse IgG+IgM (H+L) at a concentration of2.4 micrograms/mL in coating buffer (0.1 M carbonate/bicarbonate buffer,pH 9.2-9.6) at 4° C. was added to the ELISA plates overnight. The plateswere washed thrice in washing buffer (PBS+0.05 percent Tween). 100microliters/well blocking buffer (5 percent milk in wash buffer) wasadded to the plate for 1 hour at room temperature and then washed thricein washing buffer. 100 microliters/well of hybridoma supernatant wasadded and the plate incubated for 1 hour at room temperature. The plateswere washed thrice with washing buffer and 1/100,000 dilution of eithergoat anti-mouse IgG or IgM horseradish peroxidase conjugate (diluted inPBS containing 5 percent milk), 100 microliters/well, was added. Afterincubating the plate for 1 hour at room temperature the plate was washedthrice with washing buffer. 100 microliters/well of TMB solution wasincubated for 1-3 minutes at room temperature. The color reaction wasterminated by adding 50 microliters/well 2M H₂SO₄ and the plate was readat 450 nm with a reference filter of 595 nm with a Perkin-Elmer HTS7000plate reader. As indicated in FIG. 1, the AR59A269.5 hybridoma secretedprimarily antibodies of the IgG isotype.

To determine the subclass of antibody secreted by the hybridoma cells,an isotyping experiment was performed using a Mouse Monoclonal AntibodyIsotyping Kit (HyCult Biotechnology, Frontstraat, Netherlands). 500microliters of buffer solution was added to the test strip containingrat anti-mouse subclass specific antibodies. 500 microliters ofhybridoma supernatant was added to the test tube, and submerged bygentle agitation. Captured mouse immunoglobulins were detected directlyby a second rat monoclonal antibody which is coupled to colloidparticles. The combination of these two proteins creates a visual signalused to analyse the isotype. The anti-cancer antibody AR59A269.5 is ofthe IgG2a, κ isotype.

After one round of limiting dilution, hybridoma supernatants were testedfor antibodies that bound to target cells in a cell ELISA assay. Onehuman breast cancer cell line, one human ovarian cancer cell line, onehuman colon cancer cell line and 1 human normal skin cell line weretested: MDA-MB-231, OVCAR-3, Lovo and CCD-27sk respectively. All celllines were obtained from the American Type Tissue Collection (ATCC;Manassas, Va.). The plated cells were fixed prior to use. The plateswere washed thrice with PBS containing MgCl₂ and CaCl₂ at roomtemperature. 100 microliters of 2 percent paraformaldehyde diluted inPBS was added to each well for 10 minutes at room temperature and thendiscarded. The plates were again washed with PBS containing MgCl₂ andCaCl₂ three times at room temperature. Blocking was done with 100microliters/well of 5 percent milk in wash buffer (PBS+0.05 percentTween) for 1 hour at room temperature. The plates were washed thricewith wash buffer and the hybridoma supernatant was added at 75microliters/well for 1 hour at room temperature. The plates were washed3 times with wash buffer and 100 microliters/well of 1/25,000dilution ofgoat anti-mouse IgG or IgM antibody conjugated to horseradish peroxidase(diluted in PBS containing 5 percent milk) was added. After 1 hourincubation at room temperature the plates were washed 3 times with washbuffer and 100 microliter/well of TMB substrate was incubated for 1-3minutes at room temperature. The reaction was terminated with 50microliters/well 2M H₂SO₄ and the plate read at 450 nm with a referencefilter of 595 nm with a Perkin-Elmer HTS7000 plate reader. The resultsas tabulated in FIG. 1 were expressed as the number of folds abovebackground compared to an in-house IgG isotype control that haspreviously been shown not to bind to the cell lines tested. Theantibodies from the hybridoma AR59A269.5 showed moderate binding to thecolon cancer cell line Lovo, followed by the ovarian cancer cell lineOVCAR-3. Binding to the other colon cancer cell line SW1116 was notdetermined (ND). AR59A269.5 displayed the lowest level of binding to thenormal skin cell line CCD-27sk.

In conjunction with testing for antibody binding the cytotoxic effect ofthe hybridoma supernatants was tested in the cell lines: MDA-MB-231,OVCAR-3, SW1116, Lovo and CCD-27sk. Calcein AM was obtained fromMolecular Probes (Eugene, Oreg.) and the assay was performed as outlinedbelow. Cells were plated before the assay at the predeterminedappropriate density. After 2 days, 75 microliters of supernatant fromthe hybridoma microtitre plates were transferred to the cell plates andincubated in a 5 percent CO₂ incubator for 5 days. The wells that servedas the positive controls were aspirated until empty and 100 microlitersof sodium azide (NaN₃, 0.01 percent, Sigma, Oakville, ON), cycloheximide(CHX, 0.5 micromolar, Sigma, Oakville, ON) or anti-EGFR antibody (c225,IgG1, kappa, 5 micrograms/mL, Cedarlane, Hornby, ON) was added. After 5days of treatment, the plates were then emptied by inverting andblotting dry. Room temperature DPBS (Dulbecco's phosphate bufferedsaline) containing MgCl₂ and CaCl₂ was dispensed into each well from amultichannel squeeze bottle, tapped 3 times, emptied by inversion andthen blotted dry. 50 microliters of the fluorescent calcein dye dilutedin DPBS containing MgCl₂ and CaCl₂ was added to each well and incubatedat 37° C. in a 5 percent CO₂ incubator for 30 minutes. The plates wereread in a Perkin-Elmer HTS7000 fluorescence plate reader and the datawas analyzed in Microsoft Excel. The results are tabulated in FIG. 1.Supernatant from the AR59A269.5 hybridoma produced specific cytotoxicityof 15 percent on the OVCAR-3 cells, which was 22 and 42 percent of thecytotoxicity obtained in the OVCAR-3 cells with the positive controlssodium azide and cycloheximide respectively. AR59A269.5 also showedspecific cytotoxicity of 46 percent on the SW1116 cells, which wasgreater than the cytotoxicity obtained in these cells withcycloheximide, and two times greater than that obtained with theantibody c225, directed against epidermal growth factor receptor.Results from FIG. 1 demonstrated that the cytotoxic effects ofAR59A269.5 were not proportional to the binding levels on the two cancercell types. Although binding was higher in Lovo than in OVCAR-3, invitro cytotoxicity was only observed in the OVCAR-3 cells in this assay.As shown in FIG. 1, AR59A269.5 did not produce cytotoxicity in theCCD-27sk normal cell line. The known non-specific cytotoxic agentscycloheximide and NaN₃ generally produced cytotoxicity as expected. Theanti-EGFR antibody c225 produced cytotoxicity as expected on SW1116.

EXAMPLE 2

In vitro Binding

AR59A269.5 monoclonal antibody was produced by culturing the hybridomain roller bottles (Corning Inc. NY, USA) and collecting the supernatantafter 10 to 14 days of incubation. Standard antibody purificationprocedures with Protein G Sepharose 4 Fast Flow (Amersham Biosciences,Baie d'Urfé, QC) were followed. It is within the scope of this inventionto utilize monoclonal antibodies that are de-immunized, humanized,chimerized or murine.

Binding of AR59A269.5 to breast (MDA-MB-231), colon (DLD-1, Lovo, SW620and SW1116), prostate (PC-3) and ovarian (OVCAR-3) cancer cell lines,and non-cancer cell lines from skin (CCD-27sk) and lung (Hs888.Lu) wasassessed by flow cytometry (FACS). All cell lines were obtained from theAmerican Type Tissue Collection (ATCC; Manassas, Va.). Cells wereprepared for FACS by initially washing the cell monolayer with DPBS(without Ca⁺⁺ and Mg⁺⁺). Cell dissociation buffer (INVITROGEN,Burlington, ON) was then used to dislodge the cells from their cellculture plates at 37° C. After centrifugation and collection, the cellswere resuspended in DPBS containing MgCl₂, CaCl₂ and 2 percent fetalbovine serum at 4° C. (staining media) and counted, aliquoted toappropriate cell density, spun down to pellet the cells and resuspendedin staining media at 4° C. in the presence of test antibodies(AR59A269.5) or control antibodies (isotype control, anti-EGFR) at 20micrograms/mL on ice for 30 minutes. Prior to the addition of AlexaFluor 546-conjugated secondary antibody the cells were washed once withstaining media. The Alexa Fluor 546-conjugated antibody in stainingmedia was then added for 30 minutes at 4° C. The cells were then washedfor the final time and resuspended in fixing media (staining mediacontaining 1.5 percent paraformaldehyde). Flow cytometric acquisition ofthe cells was assessed by running samples on a FACSarray™ using theFACSarray™ System Software (BD Biosciences, Oakville, ON). The forward(FSC) and side scatter (SSC) of the cells were set by adjusting thevoltage and amplitude gains on the FSC and SSC detectors. The detectorsfor the fluorescence (Alexa-546) channel was adjusted by runningunstained cells such that cells had a uniform peak with a medianfluorescent intensity of approximately 1-5 units. For each sample,approximately 10,000 gated events (stained fixed cells) were acquiredfor analysis and the results are presented in FIG. 2.

FIG. 2 tabulated the mean fluorescence intensity fold increase aboveisotype control. Representative histograms of AR59A269.5 antibodies werecompiled for FIG. 3. AR59A269.5 showed strongest binding to the coloncancer cell lines DLD-1 (192.9-fold), Lovo (109.1-fold), and SW620(138.3-fold). Strong binding was also observed with the SW1116 coloncancer cell line (44.2-fold), the ovarian cancer line OVCAR-3(93.7-fold) and the prostate cancer cell line PC-3 (31.6-fold). Weakerbinding was seen with the breast cancer cell line MDA-MB-231 (5.3-fold).AR59A269.5 did not bind to the normal lung and skin cell lines, Hs888.Luand CCD-27sk, respectively. These data demonstrate that AR59A269.5 bindsto different human cancer cell lines with the highest degree of bindingto colon cancer. AR59A269.5 also demonstrated selectively in bindingsince no detectable binding was detected to both human normal celllines. These results are consistent with those obtained in Example 1.

EXAMPLE 3

In Vivo Tumor Experiments with Lovo Cells

Examples 1 and 2 demonstrated that AR59A269.5 had anti-cancer propertiesagainst colon and ovarian cancer cell lines and demonstrated the highestbinding to colon cell types. The antibody was then tested in an in vivomodel of human colon cancer. With reference to FIGS. 4 and 5, 4 to 6week old female SCID mice were implanted with 1 million human coloncancer cells (Lovo) in 100 microlitres saline injected subcutaneously inthe scruff of the neck. The mice were randomly divided into 2 treatmentgroups of 5. On the day after implantation, 20 mg/kg of AR59A269.5 testantibody or buffer control was administered intraperitoneally to eachcohort in a volume of 300 microlitres after dilution from the stockconcentration with a diluent that contained 2.7 mM KCl, 1 mM KH₂PO₄, 137mM NaCl and 20 mM Na₂HPO₄. The antibody and control samples were thenadministered once per week for a period of 7 weeks in the same fashion.Tumor growth was measured about every seventh day with calipers for upto 8 weeks or until individual animals reached Canadian Council forAnimal Care (CCAC) endpoints. Body weights of the animals were recordedonce per week for the duration of the study. At the end of the study allanimals were euthanised according to CCAC guidelines.

AR59A269.5 prevented tumor growth and reduced tumor burden in apreventative in vivo model of human colon cancer. On day 49post-implantation, 1 day before the last treatment dose, the mean tumorvolume in the AR59A269.5 treated group was 38 percent of the tumorvolume in the buffer control-treated group (p=0.0335, t-test, FIG. 4).

There were no clinical signs of toxicity throughout the study. Bodyweight measured at weekly intervals was used as a surrogate forwell-being and failure to thrive. As seen in FIG. 5, there was nosignificant difference in body weight between the groups at the end ofthe treatment period (p=0.2391, t-test). Within each group, the averagebody weight of the animals did not vary significantly between the startand the end of the study period (buffer control-treated group p=0.0752,t-test; AR59A269.5-treated group p=0.4234).

Therefore AR59A269.5 was well-tolerated and decreased the tumor burdenin a human colon cancer xenograft model.

EXAMPLE 4

In Vivo Tumor Experiments with DLD-1 Cells

Results from Example 3 were extended to a different model of human coloncancer. With reference to FIGS. 6 and 7, 4 to 6 week old female SCIDmice were implanted with 5 million human colon cancer cells (DLD-1) in100 microlitres saline injected subcutaneously in the scruff of theneck. The mice were randomly divided into 2 treatment groups of 5. Onthe day after implantation, 20 mg/kg of AR59A269.5 test antibody orbuffer control was administered intraperitoneally to each cohort in avolume of 300 microlitres after dilution from the stock concentrationwith a diluent that contained 2.7 mM KCl, 1 mM KH₂PO₄, 137 mM NaCl and20 mM Na₂HPO₄. The antibody and control samples were then administeredonce per week for the duration of the study in the same fashion. Tumorgrowth was measured about every seventh day with calipers. The study wascompleted after 7 injections (48 days), as the animals reached CCACend-points due to large ulcerated lesions. Body weights of the animalswere recorded once per week for the duration of the study. At the end ofthe study all animals were euthanized according to CCAC guidelines.

AR59A269.5 prevented tumor growth and reduced tumor burden in apreventative in vivo model of human colon cancer. On day 48post-implantation, 5 days after the last treatment dose, the mean tumorvolume in the AR59A269.5 treated group was 19 percent of the tumorvolume in the buffer control-treated group (p=0.001, t-test, FIG. 6).

There were no clinical signs of toxicity throughout the study. Bodyweight measured at weekly intervals was a surrogate for well-being andfailure to thrive. As seen in FIG. 7, there was no significantdifference in body weight between the control-treated group and theAR59A269.5 treated group over the course of the study. Within groups,the average body weight of the animals did not vary significantly overthe course of the study.

Therefore AR59A269.5 was well-tolerated and decreased the tumor burdenin two different colon cancer xenograft models.

EXAMPLE 5

Human Tumor Xenograft Staining

IHC studies were conducted to characterize the AR59A269.5 antigendistribution in human xenografts. IHC optimization studies showed thatthe antibody does not bind to formalin fixed tissues but does bind tofrozen tissue sections. Tumor xenografts were harvested from SCID micehoused at the animal facility laboratory at the Toronto GeneralHospital. Mice were injected with either the colon cancer cell lineSW1116 or the breast cancer cell line MDA-MB-231. Cell lines wereobtained from the American Type Tissue Collection (ATCC; Manassas, Va.).Tumors were immersed in OCP embedding gel and snapped frozen in nitrousoxide. Tissue blocks were sent to the Pathology research programlaboratory at the Toronto General Hospital for processing.

Tissue sections were transferred from −80° C. to −20° C. and after 1hour the sections were fixed for 10 minutes in cold (−20° C.) acetoneand allowed to come to room temperature. The slides were then rinsed 3times for 2 minutes each in cold (4° C.) PBS. The slides were thenimmersed in 3 percent hydrogen peroxide solution for 10 minutes, washedwith PBS 3 times for 5 minutes each, dried and then incubated withUniversal blocking solution (Dako, Toronto, Ontario) for 5 minutes atroom temperature. AR59A269.5 was diluted in antibody dilution buffer(Dako, Toronto, Ontario) to its working concentration (5 micrograms/mL).Cytokeratin-7 was used as a positive antibody control (ready to use;Dako, Toronto, Ontario) and antibody dilution buffer (Dako, Toronto,Ontario) was used as a negative control. The primary antibodies wereincubated for 1 hour at room temperature. The slides were washed withPBS 3 times for 5 minutes each. Immunoreactivity of the primaryantibodies was detected/visualized with HRP conjugated secondaryantibodies (ready to use; Dako Envision System, Toronto, Ontario) for 30minutes at room temperature. Following this step the slides were washedwith PBS 3 times for 5 minutes each and a color reaction was developedby adding DAB (3,3′-diaminobenzidine tetrahydrachloride, Dako, Toronto,Ontario) chromogen substrate solution for immunoperoxidase staining for10 minutes at room temperature. Washing the slides in tap waterterminated the chromogenic reaction. Following counterstaining withMeyer's Hematoxylin (Sigma Diagnostics, Oakville, ON), the slides weredehydrated with graded ethanols (75-100%) and cleared with xylene. Usingmounting media (Dako, Toronto, Ontario) the slides were coverslipped.Slides were microscopically examined using an Axiovert 200 (ZiessCanada, Toronto, ON) and digital images acquired and stored usingNorthern Eclipse Imaging Software (Mississauga, ON). Results were read,scored and interpreted by a histopathologist.

Binding of AR59A269.5 to human xenografts (colon cancer SW1116 andbreast cancer MDA-MB-231) showed strong binding to SW1116 and weakerbinding to MDA-MB-231 (FIG. 8). The binding was restricted to tumorcells. The cellular localization was membranous in SW1116 andcytoplasmic membranous in MDA-MB-231with a diffuse staining pattern(FIG. 9). The percentage of positive cells was more than 50 percent forSW1116 and less than 50 percent for MDA-MB-231. These results confirmwhat was observed by FACS in Example 2 for these cell lines in vitro anddemonstrates that expression of the antigen is maintained in vivo as axenograft.

The preponderance of evidence shows that AR59A269.5 mediates anti-cancereffects through ligation of an epitope present on cancer cell lines andhuman tumor tissue. Further it could be shown that the AR59A269.5antibody could be used in detection of cells and/or tissues whichexpress the epitope which specifically binds thereto, utilizingtechniques illustrated by, but not limited to FACS, cell ELISA or IHC.

All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention isillustrated, it is not to be limited to the specific form or arrangementof parts herein described and shown. It will be apparent to thoseskilled in the art that various changes may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is shown and described in the specification.One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Anyoligonucleotides, peptides, polypeptides, biologically relatedcompounds, methods, procedures and techniques described herein arepresently representative of the preferred embodiments, are intended tobe exemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the appended claims. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. The isolated monoclonal antibody produced by the hybridoma depositedwith the IDAC as accession number 170505-01.
 2. A humanized antibodyproduced from the isolated monoclonal antibody of claim
 1. 3. A chimericantibody produced from the isolated monoclonal antibody of claim
 1. 4.The isolated hybridoma cell line deposited with the IDAC as accessionnumber 170505-01.
 5. A method for initiating antibody induced cellularcytotoxicity of cancerous cells in a tissue sample selected from a humantumor comprising: providing a tissue sample from said human tumor;providing the isolated monoclonal antibody produced by the hybridomadeposited with the IDAC as accession number 170505-01 or a CDMABthereof, which CDMAB is characterized by an ability to competitivelyinhibit binding of said isolated monoclonal antibody to its targetantigen; and contacting said isolated monoclonal antibody or CDMABthereof with said tissue sample; wherein binding of said isolatedmonoclonal antibody or CDMAB thereof with said tissue sample inducescellular cytotoxicity.
 6. A CDMAB of the isolated monoclonal antibody ofclaim
 1. 7. A CDMAB of the humanized antibody of claim
 2. 8. A CDMAB ofthe chimeric antibody of claim
 3. 9. The isolated antibody or CDMABthereof, of any one of claims 1, 2, 3, 6, 7 or 8 conjugated with amember selected from the group consisting of cytotoxic moieties,enzymes, radioactive compounds, and hematogenous cells.
 10. A bindingassay to determine a presence of cancerous cells in a tissue sampleselected from a human tumor, which is specifically bound by the isolatedmonoclonal antibody produced by hybridoma cell line AR59A269.5 havingIDAC Accession No. 170505-01, comprising: providing a tissue sample fromsaid human tumor; providing at least one isolated monoclonal antibody orCDMAB thereof that recognizes the same epitope or epitopes as thoserecognized by the isolated monoclonal antibody produced by a hybridomacell line AR59A269.5 having IDAC Accession No. 170505-01; contactingsaid at least one isolated monoclonal antibody or CDMAB thereof withsaid tissue sample; and determining binding of said at least oneisolated monoclonal antibody or CDMAB thereof with said tissue sample;whereby the presence of said cancerous cells in said tissue sample isindicated.
 11. A method of treating a human tumor in a mammal, whereinsaid human tumor expresses an at least one epitope of an antigen whichspecifically binds to the isolated monoclonal antibody encoded by aclone deposited with the IDAC as accession number 170505-01 or a CDMABthereof, which CDMAB is characterized by an ability to competitivelyinhibit binding of said isolated monoclonal antibody to its targetantigen, comprising administering to said mammal said monoclonalantibody or CDMAB thereof in an amount effective to result in areduction of said mammal's tumor burden.
 12. The method of claim 11wherein said isolated monoclonal antibody is conjugated to a cytotoxicmoiety.
 13. The method of claim 12 wherein said cytotoxic moiety is aradioactive isotope.
 14. The method of claim 11 wherein said isolatedmonoclonal antibody or CDMAB thereof activates complement.
 15. Themethod of claim 11 wherein said isolated monoclonal antibody or CDMABthereof mediates antibody dependent cellular cytotoxicity.
 16. Themethod of claim 11 wherein said isolated monoclonal antibody ishumanized.
 17. The method of claim 11 wherein said isolated monoclonalantibody is chimerized.
 18. A method of treating a human tumorsusceptible to antibody induced cellular cytotoxicity in a mammal,wherein said human tumor expresses at least one epitope of an antigenwhich specifically binds to the isolated monoclonal antibody encoded bya clone deposited with the IDAC as accession number 170505-01 or a CDMABthereof, which CDMAB is characterized by an ability to competitivelyinhibit binding of said isolated monoclonal antibody to its targetantigen, comprising administering to said mammal said monoclonalantibody or said CDMAB thereof in an amount effective to result in areduction of said mammal's tumor burden.
 19. The method of claim 18wherein said isolated monoclonal antibody is conjugated to a cytotoxicmoiety.
 20. The method of claim 19 wherein said cytotoxic moiety is aradioactive isotope.
 21. The method of claim 18 wherein said isolatedmonoclonal antibody or CDMAB thereof activates complement.
 22. Themethod of claim 18 wherein said isolated monoclonal antibody or CDMABthereof mediates antibody dependent cellular cytotoxicity.
 23. Themethod of claim 18 wherein said isolated monoclonal antibody ishumanized.
 24. The method of claim 18 wherein said isolated monoclonalantibody is chimerized.